Influence of genetic knockout of Pept2 on the in vivo disposition of endogenous and exogenous carnosine in wild-type and Pept2 null mice.

Carnosine (beta-alanyl-l-histidine), an endogenous dipeptide substrate of the proton-coupled oligopeptide transporter PEPT2, plays an important role in many physiological processes. This study examined the effect of PEPT2 on the disposition of endogenous and exogenous carnosine in wild-type and Pept2 null mice. After exogenous dosing of [(3)H]carnosine (1 nmol/g iv bolus), a marked increase was observed in its systemic clearance in Pept2 null mice (0.50 vs. 0.29 ml/min), resulting in a decreased systemic exposure of dipeptide (area under the curve = 43.7 vs. 73.0 microM). Carnosine uptake was substantially reduced in the kidney of Pept2 null mice, and renal clearance increased 18-fold in this genotype (206 vs. 11.5 microl/min). Fractional reabsorption of carnosine in Pept2 null mice was only one-fifth that in wild-type animals (0.20 vs. 0.94). PEPT2 also had a substantial impact in brain where the cerebrospinal fluid (CSF)-to-plasma concentration ratio of carnosine was eightfold greater in Pept2 null mice (0.70 vs. 0.08). With respect to endogenous carnosine levels, significant reductions were observed in Pept2 null compared with wild-type mice for choroid plexus (0.026 vs. 0.20 mmol/kg), olfactory bulb (1.12 vs. 1.79 mmol/kg), and spleen (0.019 vs. 0.029 mmol/kg). In contrast, carnosine levels in the skeletal muscle of Pept2 null mice were significantly increased (1.70 vs. 1.14 mmol/kg), and no differences were observed between genotypes for endogenous carnosine levels in plasma and CSF. These results demonstrate that PEPT2 significantly modulates the disposition of exogenous carnosine. However, endogenous carnosine levels may be under homeostatic control to maintain systemic and central concentrations under physiological in vivo conditions.

A novel, quantitative assay for homocarnosine in cerebrospinal fluid using stable-isotope dilution liquid chromatography-tandem mass spectrometry.

We describe a rapid and sensitive method for the quantification of homocarnosine in physiological fluids, with particular emphasis on cerebrospinal fluid (CSF). Homocarnosine was quantified as the butyl derivative, with (2)H(2)-l-homocarnosine as internal standard. Following deproteinization of CSF samples, supernatants were evaporated to dryness and derivatized with 10% 6M HCl in butanol. Samples were chromatographed on a C(18) column and detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS) operating in the multiple reaction monitoring mode. The intra- and inter-assay variations were 4.6 and 10.9%, respectively. Mean recovery of homocarnosine at two concentrations was 105%. The limit of detection in CSF approximated 20 nmol/L. CSF homocarnosine is age dependent and ranges from <0.02 to 10 micromol/L. Our method is applicable to the analysis of CSF derived from patients with heritable defects in the GABA pathway, patients with homocarnosinosis or serum carnosinase deficiency, and should be applicable to other model systems in order to further explore the biological role and significance of homocarnosine in mammalian systems.

Amino acid levels in the cerebrospinal fluid of newly diagnosed epileptic patients: effect of vigabatrin and carbamazepine monotherapies.

We studied the CSF amino acid levels of 42 patients with newly diagnosed epilepsy before treatment with antiepileptic medication and during monotherapy with either vigabatrin or carbamazepine. The present study shows that patients with newly diagnosed epilepsy have elevated levels of the excitatory amino acid glutamate in CSF. Vigabatrin monotherapy effectively prevents the appearance of seizures in patients with high baseline CSF glutamate levels. In these patients, vigabatrin not only elevates the levels of gamma-aminobutyric acid, but also decreases the elevated levels of glutamate in CSF, which may also be important to the antiepileptic efficacy of vigabatrin. Patients with low CSF glutamate levels did not benefit from vigabatrin-induced changes in amino acid levels and successful monotherapy with carbamazepine did not affect CSF amino acid levels. The elevation of gamma-aminobutyric acid is thus not the only way to achieve seizure control and there are several factors underlying the generation and control of seizures. Follow-up of the patients with high baseline glutamate CSF levels will show if the observed abnormalities are related to the severity of epilepsy in individual patients and if early treatment with vigabatrin of these patients could prevent the development of intractable epilepsy.

Effect of long-term vigabatrin therapy on selected neurotransmitter concentrations in cerebrospinal fluid.

Ten patients, suffering from drug-resistant complex partial seizures were treated for a period of up to 3 years with vigabatrin (Sabril). Vigabatrin is a novel antiepileptic agent, whose action is based on the inhibition of gamma-aminobutyric acid (GABA) aminotransferase, the enzyme responsible for the catabolism of the neurotransmitter GABA. Samples of lumbar cerebrospinal fluid were obtained from the patients prior to commencing vigabatrin therapy, and thereafter at 6 months, 1 year, 2 years, and up to 3 years following the initiation of vigabatrin treatment. The influence of vigabatrin on the cerebrospinal fluid concentrations of free and total GABA, homocarnosine, homovanillic acid, 5-hydroxyindoleacetic acid, and 3-methoxy-4-hydroxyphenylethylene glycol, as well as of the drug itself, was assessed. All patients demonstrated a clinical response to vigabatrin, and the drug was well tolerated over the entire observation period. Mean (+/- SD) reduction of seizure frequency was 65% +/- 23% (range, 26% to 100%) when comparing the end of the treatment period to the previgabatrin baseline. The cerebrospinal fluid concentrations of both free and total GABA and of the dipeptide homocarnosine showed approximately 2- to 5-fold increases over baseline values, with free GABA and homocarnosine being the more sensitive variables. Cerebrospinal fluid concentrations of homovanillic acid, 5-hydroxyindoleacetic acid, and 3-methoxy-4-hydroxyphenylethylene glycol were not altered in a significant manner over the observation period. These findings support the concept that the effects of vigabatrin are restricted to an effect on GABA catabolism and do not extend to the neurotransmitters dopamine and norepinephrine. Clinical efficacy and elevation of GABA and homocarnosine concentration were sustained over the period of observation.

Cerebrospinal fluid parameters in healthy volunteers during serial lumbar punctures.

Lumbar punctures were performed on four occasions over a 5-day period (8:30 a.m. on days 1, 3, and 5; 2:30 p.m. on day 2) on 10 normal volunteers (five of each sex; mean age, 27.7 years) to assess, with repeated sampling, the day-to-day variation of selected CSF parameters. Two subjects abstained from the lumbar puncture on day 5 due to headache after the third puncture. Lumbar CSF was analyzed for concentrations of free and total gamma-aminobutyric acid (GABA), homocarnosine, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA), total protein, albumin, and immunoglobulin (Ig)G. No significant concentration differences were found between the afternoon and next morning samples. No differences were found in concentrations of free GABA, total GABA, homocarnosine, 5-HIAA, or albumin across the study. In contrast, HVA concentrations significantly increased by day 5, whereas total protein and IgG decreased during the study. The most likely explanation for these changes involves the known concentration gradients in the CSF column.

Inhibitory and excitatory amino acids in cerebrospinal fluid of chronic epileptic patients.

We studied the levels of excitatory and inhibitory amino acids in the cerebrospinal fluid (CSF) of 28 epileptic patients (24 with partial type seizures, 4 with primary generalized seizures) and 12 controls. The levels of aspartate were 63% (p less than 0.01), glutamine 129% (p less than 0.001), and homocarnosine 127% (p less than 0.005) that of controls. The concentrations of glutamate, asparagine, total GABA, free GABA, taurine, and glycine did not differ between epileptic patients and controls. Patients with partial epilepsy had a pattern of amino acids in CSF similar to that in patients with primary generalized seizures. In the present study we did not observe increased excitation or decreased inhibition in the seizure-active brains of epileptics, as far as the CSF levels of amino acids reflect their levels in the brain.

Cerebrospinal fluid GABA and seizure control with vigabatrin.

1. To evaluate the relationship between the clinical response and enhancement of GABAergic neurotransmission, for 6 months we administered vigabatrin (gamma-vinyl-GABA, GVG) to 75 patients with complex partial epilepsy. Total GABA (TGABA), free GABA (FGABA), homocarnosine (HC), and GVG concentrations were measured in CSF of these patients before and during GVG treatment. 2. Over 50% reduction in seizures was found in 55% of the patients. Dose-reduction resulted in a relapse, i.e. the return of seizures. 3. At baseline TGABA, FGABA, and HC did not differ in responders and nonresponders. After GVG treatment, the TGABA and HC levels were lower in nonresponders (P less than 0.001), but the GVG and FGABA levels did not differ. The GVG dose reduction resulted in a concomitant decrease in TGABA, FGABA, HC and GVG (P less than 0.001). 4. According to our results GVG is an effective anticonvulsant drug in complex partial seizures. In nonresponders the poor anticonvulsant response may be related to the lower elevation of the CSF markers of GABAergic neuronal activity in this group compared with the responders.

Levels of total gamma-aminobutyric acid (GABA), free GABA and homocarnosine in cerebrospinal fluid of epileptic patients before and during gamma-vinyl-GABA (vigabatrin) treatment.

Levels of total gamma-aminobutyric acid (TGABA), free GABA (FGABA), and homocarnosine (HC) were studied in CSF taken from 12 controls and 28 patients with drug-refractory epilepsy before and during 7 months of gamma-vinyl-GABA (GVG) administration. At baseline TGABA and FGABA in CSF of epileptic patients did not differ from that of the controls. In epileptic patients HC was 127% of that in controls. During GVG treatment TGABA was 283%, FGABA 197%, and HC 310% of the levels at baseline in the same patients. The patients who had over 50% reduction in seizure frequency during GVG (responders, 46% of the study population) at baseline had higher TGABA and HC in CSF than patients with less than 50% reduction in seizures (non-responders). During GVG the responders and nonresponders had similar levels of different GABAergic markers. The present study shows that in man GVG treatment effectively suppresses seizures in nearly half of the epileptic patients who had previously been drug-refractory. The elevated levels of GABAergic markers in CSF are not, however, necessarily related to good seizure control during GVG.

Effects of single doses of vigabatrin on CSF concentrations of GABA, homocarnosine, homovanillic acid and 5-hydroxyindoleacetic acid in patients with complex partial epilepsy.

Vigabatrin, as a single oral dose of 50 mg/kg, was administered to 11 patients with drug-refractory complex partial epilepsy. Serial lumbar punctures were performed prior to and 5 times within the first week following treatment. Cerebrospinal fluid (CSF) concentrations of total GABA, free GABA, homocarnosine, homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and vigabatrin were determined as well as blood vigabatrin levels. CSF GABA, homocarnosine, HVA and 5-HIAA concentrations increased by 6 h after the single dose and remained elevated for up to 5-7 days. In contrast, CSF and blood vigabatrin levels were maximal within the first 24 h and were no longer detectable thereafter. Hence, these results are consistent with vigabatrin acting as an irreversible inhibitor of GABA-transaminase and suggest that it may also increase biogenic amine turnover.

Clinical and biochemical effects of gamma-vinyl Gaba in tardive dyskinesia.

Clinical and biochemical effects of Gamma-vinyl-Gaba (GVG) have been evaluated in a blind video-controlled study in 10 psychiatric patients (mean age 71 yr) with tardive dyskinesia. CSF free and total Gaba and homocarnosine concentrations increased from three to five fold with GVG treatment. Despite the GVG-induced biological effects on Gaba metabolism, GVG did not consistently improve tardive dyskinesia. Psychomotor side-effects occurred in older patients, who only tolerated GVG dosages of 2-4 g/day.

2-Pyrrolidinone in human cerebrospinal fluid: a major constituent of total gamma-aminobutyric acid.

2-Pyrrolidinone, the lactam of gamma-aminobutyric acid (GABA), is identified as the major constituent of total GABA in human CSF. Structural elucidation was done by mass spectrometry. In lumbar CSF of four patients, 2-pyrrolidinone represented about 54% of GABA found after acid hydrolysis, thus accounting for essentially all of the hitherto unknown GABA fraction in CSF.

Cerebrospinal fluid GABA and homocarnosine concentrations in patients with Friedreich's ataxia, Parkinson's disease, and Huntington's chorea.

Free and total gamma-aminobutyric acid (GABA) and homocarnosine concentrations were measured in the lumbar cerebrospinal fluid (CSF) of patients with Friedreich's ataxia, Huntington's chorea, and Parkinson's disease (with and without levodopa treatment), and compared with those determined in control subjects. Values found in Friedreich's ataxia or Parkinson's disease were not significantly different from those in controls. Unexpectedly, in Huntington patients, known to have a characteristic decrease in GABA concentrations in specific brain areas, CSF concentrations of total GABA and homocarnosine were significantly higher, whereas free GABA was not different from controls. These findings indicate that the measurement of CSF GABA and homocarnosine in patients with CNS degenerative diseases should be interpreted cautiously.

Homocarnosinosis: influence of dietary restriction of histidine.

Homocarnosinosis, an inherited disorder, is characterized by an elevated level of the dipeptide homocarnosine (Hca) in the CSF and the brain and, in addition, by carnosinuria and serum carnosinase deficiency. In three children with homocarnosinosis the biochemical aberration co-exists with paraplegia, retinitis pigmentosa, and a progressive mental deficiency. In the mother, however, only the biochemical aberration was present without clinical symptoms. In order to study whether this elevated level of Hca and increased excretion of carnosine (Car) could be reduced towards normal, a dietary regiment with restriction of histidine (His) was maintained for nearly 2 1/2 years for two of the patients, 33 and 39 years old, with homocarnosinosis associated with neurological symptoms. His was reduced by about 90% in the CSF, in the plasma and in the urine. Within 5-6 months CSF Hca was reduced by about 70%, and urinary Car by 22 and 42%. The clinical neurological symptoms, however, did not alter significantly together with these biochemical changes.

Gamma-aminobutyric acid-transaminase deficiency: a newly recognized inborn error of neurotransmitter metabolism.

Cerebrospinal fluid aminoacid analysis in a girl with severe psychomotor retardation, hypotonia, hyperreflexia and growth acceleration showed highly increased levels of free gamma-aminobutyric acid (4.8 mumol/l; range in twenty controls 0.04-0.12, median 0.08), homocarnosine, a dipeptide of gamma-aminobutyric acid and histidine (23.4 mumol/l; control range 4.0-8.7, median 7.6) and of beta-alanine, an alternative substrate for gamma-aminobutyric acid-transaminase (0.48 mumol/l; control range 0.02-0.06, median 0.05). Liver gamma-aminobutyric acid-transaminase activity was deficient (0.07 mumol/mg protein h; range in ten controls 0.31-0.69, median 0.38). Fasting plasma growth hormone levels were increased (7.9-38.4 ng/ml; nl less than 5). Brain evoked responses were suggestive of leukodystrophy. A brother of this patient, showing a similar clinical picture, had died at one year. Postmortem examination of his brain showed leukodystrophy of the type seen in amino acidopathies such as phenylketonuria. This appears to be the first report of gamma-aminobutyric acid-transaminase deficiency.

Homocarnosinosis: lack of serum carnosinase is the defect probably responsible for elevated brain and CSF homocarnosine.

Patients afflicted with homocarnosinosis have elevated concentrations of homocarnosine in brain and CSF. It has been reported that they lack brain homocarnosinase. However, we have found that these patients are deficient in serum carnosinase, a dipeptidase which hydrolyzes homocarnosine about 5% as rapidly as it splits carnosine. Homocarnosinase could not be detected in normal human brain extracts after isoelectric focusing or DEAE-cellulose column chromatography. The ability of brain extracts to hydrolyze homocarnosine thus appears to be attributable solely to the serum carnosinase which is present because of serum trapped in the brain sample. Preliminary data indicate that homocarnosinase is probably not present in 13 other human tissues. Normal CSF contained serum carnosinase, whereas the CSF of a homocarnosinosis patient was lacking this enzyme. Thus it appears that the elevated concentrations of homocarnosine in the CSF of homocarnosinosis patients are attributable to serum carnosinase deficiency.

Total GABA and homocarnosine in CSF as indices of brain GABA concentrations.

Treatment of rats with gamma-vinyl-GABA, an inhibitor of GABA-transaminase, caused a dose- and time-related increase in brain GABA concentrations concomitant with increases in the concentrations of total GABA and homocarnosine in the CSF. At 18 h after treatment both CSF parameters correlated significantly with brain GABA concentrations. However, only total GABA in CSF accurately reflected brain GABA concentrations as a function of time after treatment and is therefore the preferred index.

Homocarnosine levels in cerebrospinal fluid of patients with infantile spasms under ACTH therapy.

The alteration of homocarnosine (dipeptide of GABA and histidine) levels in cerebrospinal fluid (CSF) was studied on 8 cases with infantile spasms under ACTH therapy. CSF was obtained by lumbar puncture before and during ACTH therapy. Homocarnosine was determined by the amino acid autoanalyzer. The attacks of infantile spasms were well controlled in all 8 cases by ACTH therapy. Homocarnosine levels in cerebrospinal fluid of each case decreased from 1.09 to 0.55, 0.92 to 0.41, 1.23 to 0.90, 0.79 to 0.49, 1.06 to 0.75, 1.06 to 0.89, 0.84 to 0.33 and 2.17 to 0.97 mumol/dl, respectively. The decrease of homocarnosine in CSF were 49.5%, 55.4%, 26.8%, 38.0%, 29.2%, 16.0%, 60.7% and 55.3%, respectively. This finding suggests that the decrease of homocarnosine in CSF may be related to the disappearance of attacks of infantile spasms.